Genomic evidence for rediploidization and adaptive evolution following the whole-genome triplication

Whole-genome duplication (WGD), or polyploidy, events are widespread and significant in the evolutionary history of angiosperms. However, empirical evidence for rediploidization, the major process where polyploids give rise to diploid descendants, is still lacking at the genomic level. Here we present chromosome-scale genomes of the mangrove tree Sonneratia alba and the related inland plant Lagerstroemia speciosa. Their common ancestor has experienced a whole-genome triplication (WGT) approximately 64 million years ago coinciding with a period of dramatic global climate change. Sonneratia, adapting mangrove habitats, experienced extensive chromosome rearrangements post-WGT. We observe the WGT retentions display sequence and expression divergence, suggesting potential neo- and sub-functionalization. Strong selection acting on three-copy retentions indicates adaptive value in response to new environments. To elucidate the role of ploidy changes in genome evolution, we improve a model of the polyploidization–rediploidization process based on genomic evidence, contributing to the understanding of adaptive evolution during climate change.

2. If S. alba and L. speciosa have shared WGT event, then why the ks peak is not the same.This need to be explained.
3. I also suggest to calculate the gene numbers between different species in HOGs obtained by orthofinder.If there were a WGT, the proportion of 1:3:3 must be much higher than 1:2:3 or something else. 4. Line 136-138, 154-155.The authors skillfully integrated a trio of methodologies encompassing synteny, Ks-base, and phylogenetic approaches to characterize the occurrence and location of the whole-genome triplication event in S. alba and L. speciosa.This strategy effectively addresses the challenge posed by varying evolutionary rates across different plant species, ensuring more robust identification of features and positions of polyploidy events.Please provide a flowchart to illustrate it and enhance clarity. 5. Line 182-194.The authors performed a chromosome-scale comparative investigation among mangrove species and its relative L. speciosa and found the mangrove genome experienced more chromosome changes.To improve the understanding of the distinct habitats, please provide additional information about the habitats of the related species.
6. Line 197-200.The authors mentioned, "During periods of dramatic global environment and climate change, newly formed polyploids typically possess a significant fitness advantage over diploids".In support of this assertion, they have illustrated the specific global environment when the WGD/WGT happened in Fig. 3a.However, these images are devoid of textual descriptions.Please provide descriptions with references that elucidate the environment prevalent during that period.7. Line 196-211.The authors propose a model elucidating the polyploidization-rediploidization process.It seems that a cyclical nature in plant genome dynamics.It prompts consideration of the current stage of this cycle within plants.What stage are plants in now?Whether there are different stages between the mangrove species and its relatives?The authors need to think about and discuss it in this section.8. Line 204-205.Please add the references to this point ("all angiosperms have undergone successive rounds of polyploidization and rediploidization process").9. Line 257-296.By conducting transcriptome analysis, the authors identified WGT-retained genes related to adaptive evolution.In addition to the presentation of figures and tables, please provide a concise summary of these identified genes, along with their sequences.It could facilitate the utilization.10.Line 400-401.What model was used in the MCMC analysis?11.Line 505.The abbreviation of "confidence intervals" should be "CIs".12. Fig. 2d looks like a partially collinear relationship.Kindly make a note of this observation in the figure legend.13.Line 880."Supplementary Table S7" should be "Supplementary Table 7".14.Please add a row of anchored gene numbers to Table 1.15. Please state the source of the pictures (a, b, c) in Supplementary Figure 3. 16.Please change "Hi-C" to "Hi-C reads" in Supplementary Table 1.
Reviewer #2: Remarks to the Author: This is a very interesting manuscript taking a novel paired rediploidisation system.It presents a unique look at the rediploidisation process over medium-scale (not recent) evolutionary time.It will be of significance to workers in several fields, from ecology and evolution to fundamental plant biology.In general Does the work supports the conclusions and claims and I have no major comments that require modification of the analysis; as far as I can ascertain, the methodology is sound.I provide more minor comments below with the intention of helping to enhance the presentation.26: 'recent': 64 million years ago is not what most in the field would call a recent polyploidy event.
Nor is it necessary for the novelty here to suggest this is recent, so I propose to strike all use of 'recent'.32: 'adaptive value' can be inferred by retention, but 'accelerated evolutionary adaptation' cannot.
34: 'we propose a model': if you say you propose a model in the abstract, say something about it other than selling the quality of your genomes that served as input data.I also note that the model in the figure is a little bit 'light'; the novelty of the 'model' is to me not great, so I would suggest stressing the new model much, to be frank.54-57: this gives the false impression that WGD is an adaptive panacea when in fact most young polyploids are not stable.This downside can be clearly seen in discussion in e.g.https://doi.org/10.3389/fevo.2018.0011758: 'Plants on the Earth': suggest rephrase to 'Plants' or some other formulation; it's awkward as is.66: 'rain forests of the sea' is puzzling to me and seems not relevant.Perhaps it's a commonly used term for these species, but seems to me a little bit misleading.Rainforests are much more speciesdiverse in my mind than mangroves.78: references 51 and 52 are introduced as examples or support for the statement that high quality genomes have provided opportunity to study genomes post WGD.Immediately following this statement the current study is introduced, but there is no suggestion what the gap was following studies 51 and 52 or what the major current innovation motivating this study in relation to them.It would be good to say here how the current study contributes something truly novel.
83-84: 'The genome sequences… evolutionary research in plants' is far too weak a statement to make any interest.Suggest rewriting the end of the intro to be more powerful, stating the specific point of this study, as it currently is very weak indeed.88: it is strange to start here with HiC.What about the sequencing of the contig assembly?There is no reference given for this, so where do I go to learn about how it was originally sequenced?90: '204.46Mb': what was the expected size based on flow or kmers?I see these data are the methods; it would be better if they were in the results here too/instead.96: 'consistent with the estimated genome size': what is that estimate?100: a sentence about how the annotation was done would be nice in the results.
107: suggest to rephrase to either 'Fewer TEs accumulate' or 'Less TE accumulation' 123: 'taxa' is plural; 'taxon' is singular, which is what I think you want here.144, 5, 7, 152, 171: 'recent': this happened at around the time of the K-Pg boundry!I would disagree with the use of the word 'recent', and besides it does no benefit to call this WGT 'recent' for your story.You don't need it.
197-8: There is evidence of increased signal of WGD at times of dramatic environmental and climate change, but there is absolutely not sufficient evidence to make such a strong statement that 'newly formed polyploids typically possess a significant fitness advantage over diploids.'Typically neopolyploids are dead due e.g. in autotetraploids to meiotic catastrophe.I am passionate about polyploids and have devoted my career to studying them and I therefore also want to say this, but this statement goes much farther than the data and must be revised.See a more balanced discussion of this in e.g. the review I reference in 54-57 above.One much more appropriate suggestion would be to say: ''newly formed polyploids can possess fitness advantages over diploids" or more accurately something like "signals of WGD persistence correlate with times of environmental and climate change, suggesting potential benefit to WGD in the face of challenges".201: 'will': here too the language is too simplistic.I would suggest 'may' and to again see the review I reference above for more balanced examples.197-202: the studies referenced here are not strong and often rather old.Again, the statements should be more moderate and less absolute and referencing various excellent reviews of this broad literature in addition to the one I suggest would be much more informative: e.g.doi.org/10.1086/700636and doi: 10.1101/gad.271072.115.265: 'functional analysis': do you mean GO analysis?This is unclear in the text.Describing this analysis better in the main text is needed.The results seem interesting, but they need to be better shown.Nor do I see a clear description of this analysis in the methods.
Between the standard GO analysis and the 'functional analysis' not well described, it seems the enriched terms are rather 'high level' and not very descriptive: e.g.'regulatoin of biosynthetic process', 'regulation of metabolic processes', 'biological regulatoin'.Can the authors better highlight the more specific GOs that are better describing particular processes or functions?268-9: these are of course very interesting pathways!298: no need to say 'highly accurate' The conclusion is much better without the last sentence to be honest.There is no need at all for such a broad statement, which reduces the impact of the rest of the nice writing.319: more details should be given on the CTAB and reference it.
Reviewer #3: Remarks to the Author: The authors sequenced and assembled genomes of two related species from the family Lythraceae.One species belongs to mangrove species, while the second species (Lagerstroemia speciosa) is not adapted to the mangrove ecosystem.Overall the paper is clearly structured and well-written, however I need to review this work in comparison with similar studies published in Nat Comm and similar journals recently.In this comparison I found the present paper to contain less novelty than some other genome-based papers published in Nat Comm.Here are some key points I considered as critical: (1) Page 3. "almost all mangrove species are currently considered diploids".Please make sure that the current interpretation is correct.I did not check the cited references, however very often species are considered diploid due to the lack of other (lower) chromosome numbers in a given taxon (e.g., genus), and thus, species of Helianthus (n=17) are or were considered to be diploid (and there are more exaples of course).
(2) Page 4, line 109… Here the authors are reporting the difference in the proportion of TEs between the two genomes, not really appreciating the fact that logically 12 centromeres will have much less repeats than 24 centromeres (Lagerstroemia).And so it is also logic to expect that the mangrove species will have smaller nuclear genome than the inland species, as it was found.This part is overall trivial and concluding that plants and the investigating species have LTR retronsposons.Where are other types of repeats, including tandem repeats?The concluding sentence is somewhat naive but logic -see above -the authors should, for example, consider that logically there are less insertions of LTR-RTs in the mangrove genome due to the lower number of genomic regions where the insertions can be tolerated (typically these are pericentromere regions: 12 versus 24).
(3) Page 5.The phylogeny.With so many genome sequenced and analyzed phylogenetically I cannot be really happy about the phylogeny including 7 species (out of 250 000 as the authors correctly report in the Introduction)!It is also unclear and not discussed if their divergence time estimates are congruent with already published datings.(4) The same page, line 144."219 syntenic block pairs comprising 3,333 gene pairs in P. granatum".And later: "suggesting that P. granatum did not experience recent polyploidy events."It remains to be explain where from the 219 syntenic block pairs in P. granatum come from.
(5) The usage of "recent" for the identified whole-genome triplication 64 mya is really funny and I did not understand why the authors actually use "recent" for this very old polyploidization event.(6) Page 6. Line 156 and following.I really did not understand where from the authors get information how the WGT helped to cope with the K-Pg global extinction and later events?If I accept this tale, then it comes even before adaptive genes are introduced, researched and discussed.(7) The same page: "If we consider only polyploidy, the haploid chromosome number would be 168 (21*2*2*2)."It is not clear whether this is related to Utricularia or Lythraceae and, second, it has to be explained where 21 comes from (I guess it could be 3 x 7 of the Gamma WGT?).(8) Page 7, 1st paragraph.To me, the fact that the ancestral genome had 8 chromosome pairs is repeated three times.This part is very vague and general -the reader can look at figure 2f but what can be seen and inferred from this karyograms?It is also not very clear that actually the inland genome has retained the ancestral number of chromosomes after the WGT, but there are chromosomal rearrangements (what types of chr.rearrangements?).Suppl fig 13: why Pemphis acidula with the duplicated ancestral genome is not mentioned and used for comparisons with P. granatum and the two species investigated in here?(9) I missed more indepth analysis how the hexaploid genome was rearranged in the two species investigated.(10) The polyploidization-diploidization cycle (Figure 3) was published by several authors (some cited, some not) and, forgive me, to call this simple and already several times published scheme as A MODEL is little too much.The authors should eliminated "this model" or modify it by acknowledging researchers who published basically the same cycle shemes earlier.(11) Expression analysis.I was really not sure how this was meant and what exactly can be concluded when the same analysis was not done for the inland species and/or another (non-)mangrove species for which comparable dataset is available.(12) Also, surprisingly, I did not get any information how the hexaploid genome has been formed and if post-polyploid gene fractionation (diploidization process in general) impacted all three subgenomes in the same or different way.

Replies to reviewers' comments point by point: Reviewer #1 (Remarks to the Author):
This study assembled the chromosome-scale genomes of two Lythraceae plants and effectively presents the process of rediploidization after recent whole-genome triplication and its potential role in adaptive evolution.The authors also propose a model of the polyploidization-rediploidization process in plants, which holds significance in understanding adaptive evolution during periods of global climate change.Furthermore, mangroves comprise an exceptionally captivating group of plants, thriving in distinct and specialized environments.Sonneratia alba is one of the most representative mangrove species.Employing this clade as a case study, the authors have performed comprehensive analyses.These analyses yield a lot of genomic evidence about rediploidization in different aspects, such as sequence and expression divergence, chromosomal evolution, selective pressures, subfunctionalization, and adaptive trait evolution.Overall, I am glad to review this nice work and it is helpful to understand plant evolution.I am pleased to recommend it for publication after revisions.
[Reply]: Thanks for the positive comments.
More comments are listed below.2. If S. alba and L. speciosa have shared WGT event, then why the ks peak is not the same.This need to be explained.
[Reply]: Thanks.We understand the reviewer's concern.Synonymous substitution rates (Ks) between paralogous genes can be influenced by divergent evolutionary or substitution rates in different plants.
While the Ks peak appears slightly different between S. alba and L. speciosa, we have employed phylogenetic approaches to pinpoint more accurate positions of the polyploidy event.Our multipronged approach allows us to overcome the challenges posed by divergent evolutionary rates in different plants, enabling the identification of more precise features and positions of polyploidy events.
The revision below is much more explicit.
[Revision]: (Line 155-156) While the Ks peak appears slightly different between S. alba and L. speciosa, we performed gene tree reconstructions of the syntenic gene groups … (Line 159-161) This multipronged approach allows us to overcome the challenges posed by divergent evolutionary rates in different plants, enabling the identification of more accurate features and positions of polyploidy events 23,68-70 .
3. I also suggest to calculate the gene numbers between different species in HOGs obtained by orthofinder.If there were a WGT, the proportion of 1:3:3 must be much higher than 1:2:3 or something else.
[Reply]: Thank you for your suggestion.We have calculated the gene numbers within different species in homologous groups (HOGs).The number of HOG (pgr:lsp:sal = 1:3:3) is 315, which is relatively small compared to the HOG with a ratio of 1:2:2 (1889).As the WGTs occurred relatively long ago, following the subsequent rediploidization process, only a limited number of homologous groups retained three full copies, with 584 in S. alba and 943 in L. speciosa.Therefore, the identification of accurate features and positions of polyploidy events necessitates a combination of synteny, Ks-based, and phylogenetic approaches to uncover traces on the genome.It is important to note that the data from HOGs may not fully reflect the features of WGT.
4. Line 136-138, 154-155.The authors skillfully integrated a trio of methodologies encompassing synteny, Ks-base, and phylogenetic approaches to characterize the occurrence and location of the whole-genome triplication event in S. alba and L. speciosa.This strategy effectively addresses the challenge posed by varying evolutionary rates across different plant species, ensuring more robust identification of features and positions of polyploidy events.Please provide a flowchart to illustrate it and enhance clarity.
[Reply and Revision]: Thank the reviewer for the helpful reminder.We have added the flowchart in the supplementary information (Supplementary Fig. 11).
5. Line 182-194.The authors performed a chromosome-scale comparative investigation among mangrove species and its relative L. speciosa and found the mangrove genome experienced more chromosome changes.To improve the understanding of the distinct habitats, please provide additional information about the habitats of the related species.
[Reply]: Thanks.We have added additional information about the habitats of the mangrove tree [Revision]: Sonneratia alba inhabits low intertidal zones of downstream estuarine systems and is one of the most pervasive and salt-tolerant mangrove species widespread in the Indo West Pacific (IWP) region.Evolving specialized structures such as pneumatophores, S. alba demonstrates its waterlogging and salt tolerance, particularly in low intertidal zones.
Lagerstroemia speciosa, the closely related inland woody plant, demonstrates adaptability across diverse habitats, including lowland rainforests, riparian areas, as well as urban and rural environments.
The cultivation of L. speciosa in gardens and urban landscapes highlights its ornamental value and widespread popularity.
6. Line 197-200.The authors mentioned, "During periods of dramatic global environment and climate change, newly formed polyploids typically possess a significant fitness advantage over diploids".In support of this assertion, they have illustrated the specific global environment when the WGD/WGT happened in Fig. 3a.However, these images are devoid of textual descriptions.Please provide descriptions with references that elucidate the environment prevalent during that period.
[Reply]: We appreciate the reviewer's comment.We have provided a detailed description of the images in the legend of Fig. 3.The revision below is much more explicit.
[Revision]: (Line 958-963) The images portray the prevalent environments during various periods.In the Early Cretaceous (around 120 Mya), an arid climate prevailed 47 .At the K-Pg boundary (around 66 Mya), the dramatic global climate change following a significant asteroid collision with Earth 48 .
During the PETM (around 55 Mya), there was a notable global temperature increase and a rise in eustatic sea levels 72 .Finally, the image depicts the current environment.stages between the mangrove species and its relatives?The authors need to think about and discuss it in this section.
[Reply]: Polyploidy events have significantly influenced the evolutionary history of angiosperms, but diploid plants currently predominate.The process of rediploidization following polyploidization is crucial for polyploids, ultimately leading to modern descendants as normal diploids cytogenetically, generating important genetic and taxonomic diversity.Considering the potential role of ploidy changes in genome evolution, we improve a model based on new genomic evidence and the previous studies.This model explains the polyploidization-rediploidization process, elucidating the adaptive evolution during global upheavals and restoration.While S. alba and L. speciosa both underwent the WGT event, they are currently diploid.
Chromosome evolution analysis reveals that the mangrove species has a reduced number of chromosomes and undergoes more chromosome rearrangements compared to L. speciosa.In fact, polyploids may face substantial disadvantages, including redundant components, gene dosage imbalance, increased replication and metabolic costs, cellular mismanagement, and a higher propensity for polyploid mitosis and meiosis to produce aneuploid cells.Despite these immediate challenges, some polyploid lineages have persevered and even thrived.We have discussed the advantages and disadvantages of polyploidy and diploidy, as well as the polyploidization-rediploidization process in the Section (Line 218-238).
8. Line 204-205.Please add the references to this point ("all angiosperms have undergone successive rounds of polyploidization and rediploidization process").
[Reply and Revision]: Thanks for your suggestion.We added the references in the revision.

Reviewer #2 (Remarks to the Author):
This is a very interesting manuscript taking a novel paired rediploidisation system.It presents a unique look at the rediploidisation process over medium-scale (not recent) evolutionary time.It will be of significance to workers in several fields, from ecology and evolution to fundamental plant biology.In general Does the work supports the conclusions and claims and I have no major comments that require modification of the analysis; as far as I can ascertain, the methodology is sound.I provide more minor comments below with the intention of helping to enhance the presentation.
[Reply]: Thanks for the positive comments.26: 'recent': 64 million years ago is not what most in the field would call a recent polyploidy event.
Nor is it necessary for the novelty here to suggest this is recent, so I propose to strike all use of 'recent'.
[Reply]: Thanks for your suggestion.We have removed the "recent" used to modify whole genome triplication (WGT) from both the main text and supplementary information.32: 'adaptive value' can be inferred by retention, but 'accelerated evolutionary adaptation' cannot.
[Reply]: Thanks for your useful suggestion.We have revised related sentences in the revision.We have now incorporated genomic evidence for rediploidization, depicting changes at both the chromosome and gene levels in Fig. 3.This revision better reflects the central theme of our paper.Refer to Fig. 3 and Line 232-238 for detailed information.54-57: this gives the false impression that WGD is an adaptive panacea when in fact most young polyploids are not stable.This downside can be clearly seen in discussion in e.g.https://doi.org/10.3389/fevo.2018.00117 [Reply]: We appreciate the reviewer's suggestion.We have revised this part to address the potential misconception.The updated content now includes the disadvantages of WGD and then presents some cases about the adaptive potential of polyploidy, especially in the context of dynamic and fluctuating environmental conditions.The revised text is pasted below.
[Revision]: (Line 47-53) Despite the challenges that have emerged since the WGD, such as stable chromosome segregation, detrimental ecological interactions with diploid progenitors, and minority cytotype exclusion 35,36 , the polyploidy events observed in plants highlight their evolutionary potential.
Experimental and simulation studies have supported the adaptive potential of polyploidy, especially in the face of dramatic and fluctuating environmental conditions [37][38][39] .Overall, polyploidy has been recognized as a major driving force behind evolutionary adaptation and diversification 4,5 .58: 'Plants on the Earth': suggest rephrase to 'Plants' or some other formulation; it's awkward as is.
[Reply and Revision]: We have replaced "Plants on the Earth" with "Plants" in the revision.Please see Line 54. 66: 'rain forests of the sea' is puzzling to me and seems not relevant.Perhaps it's a commonly used term for these species, but seems to me a little bit misleading.Rainforests are much more speciesdiverse in my mind than mangroves.
[Reply]: We appreciate the reviewer's perspective, and we have removed the phrase "known as the 'rainforests of the sea'" as suggested.Please see Line 62. [Reply]: WGD events have played a significant role in the evolutionary history of angiosperms, and it has been proposed that these events contribute to plant adaptation and survival in the face of environmental changes.However, empirical evidence for rediploidization has been lacking, particularly at the genomic scale.By reconstructing ancestral genomes and inferring the trajectory of plant genome evolution, our study innovatively explores the rediploidization process following polyploidization on a genomic scale.In the revision, we have added a sentence to connect them.We appreciate the reviewer's suggestion.
[Revision]: (Line 71-74) With advancements in genome sequencing and assembly technologies, highquality chromosome-scale genomes provided an opportunity to reconstruct ancestral genomes and infer the trajectory of plant genome evolution 60,61 .We can now explore the process of rediploidization following polyploidization on a genomic scale.In this study, … 83-84: 'The genome sequences… evolutionary research in plants' is far too weak a statement to make any interest.Suggest rewriting the end of the intro to be more powerful, stating the specific point of this study, as it currently is very weak indeed.
[Reply]: In the revision, we have rephrased the end of the introduction to highlight the central theme of the manuscript.
[Revision]: (Line 77-79) Through comprehensive analyses, we trace the evolutionary history of genomes and investigate the polyploidization-rediploidization process and its implications for adaptive evolution in the face of global climate change.
88: it is strange to start here with HiC.What about the sequencing of the contig assembly?There is no reference given for this, so where do I go to learn about how it was originally sequenced?
[Reply]: Thank you for the comments.We did not explain the assembly clearly.To address this, we have included additional information and a reference regarding the contig assembly of S. alba.
[Revision]: (Line 83-86) We first utilized high-throughput chromosome conformation capture (Hi-C) technology to improve the genome of S. alba.This improvement builds upon our prior study utilizing PacBio Single-Molecule Real-Time (SMRT) sequencing and Illumina short reads sequencing 54 , resulting in a chromosome-scale assembly (Supplementary Table 2).it would be better if they were in the results here too/instead.
[Reply]: In our previous study, we assembled the genome of S. alba using PacBio SMRT sequencing and Illumina short-read sequencing.The assembled genome size is 207.20 Mb, consistent with the genome size estimated by k-mer-based analysis (211.67 Mb).After eliminating a small number of redundant sequences and anchoring to chromosome scale, the newly assembled genome derived from anchored contigs measured 204.46 Mb. 100: a sentence about how the annotation was done would be nice in the results.
[Reply]: Thank.We have added sentences about the annotation in the revision.
[Revision]: (Line 97-102) The gene prediction process involved a comprehensive approach, combining ab initio, homology-based and RNA-seq-assisted strategies.The integration of these predictions through EvidenceModeler resulted in the identification of non-redundant and consensus gene models for the S. alba and L. speciosa genomes (see Methods for details).This unveiled a total of 25,284 (Supplementary Fig. 5) and 30,497 (Supplementary Fig. 6) protein-coding genes, respectively, characterized by high completeness (Supplementary Table 3).107: suggest to rephrase to either 'Fewer TEs accumulate' or 'Less TE accumulation' [Reply]: We have revised the text based on your recommendation.
[Revision]: (Line 108) Less TE accumulation in the mangrove 123: 'taxa' is plural; 'taxon' is singular, which is what I think you want here.
[Reply and Revision]: Thanks for your comment.We have utilized "taxon" instead of "taxa" in the revision.Please see Line 125.144, 5, 7, 152, 171: 'recent': this happened at around the time of the K-Pg boundry!I would disagree with the use of the word 'recent', and besides it does no benefit to call this WGT 'recent' for your story.
You don't need it.
[Reply]: Thanks for your suggestion.We have removed the "recent" used to modify whole genome triplication (WGT) from both the main text and supplementary information.
197-8: There is evidence of increased signal of WGD at times of dramatic environmental and climate change, but there is absolutely not sufficient evidence to make such a strong statement that 'newly formed polyploids typically possess a significant fitness advantage over diploids.'Typically neopolyploids are dead due e.g. in autotetraploids to meiotic catastrophe.I am passionate about polyploids and have devoted my career to studying them and I therefore also want to say this, but this statement goes much farther than the data and must be revised.See a more balanced discussion of this in e.g. the review I reference in 54-57 above.One much more appropriate suggestion would be to say: ''newly formed polyploids can possess fitness advantages over diploids" or more accurately something like "signals of WGD persistence correlate with times of environmental and climate change, suggesting potential benefit to WGD in the face of challenges".
[Reply]: We agree with the reviewer's viewpoint.We have revised this sentence by the suggestion.
[Revision]: (Line 219-222) During periods of dramatic global environment and climate change, newly formed polyploids can possess fitness advantages over diploids.This is supported by evidence that the persistence of WGD correlates with times of environmental and climate change, suggesting potential benefit for the WGD in the face of challenges 4,35,45,[74][75][76][77] .201: 'will': here too the language is too simplistic.I would suggest 'may' and to again see the review I reference above for more balanced examples.
[Reply]: We have revised this sentence based on your recommendation.
[Revision]: (Line 226-228) As climatic conditions stabilize and environmental conditions improve, polyploids may experience reduced fitness compared to diploids due to the accumulation of genetic load, increased mutational load, slower positive selection, and reduced growth rates 35,37,81,82 .197-202: the studies referenced here are not strong and often rather old.Again, the statements should be more moderate and less absolute and referencing various excellent reviews of this broad literature in addition to the one I suggest would be much more informative: e.g.doi.org/10.1086/700636and doi: 10.1101/gad.271072.115.
[Reply]: Thanks for your suggestion.Incorporating the literature you kindly provided, we have revised the text to provide a more comprehensive and balanced perspective on the advantages and disadvantages of polyploidy and diploidy.
[Revision]: (Line 219-228) During periods of dramatic global environment and climate change, newly formed polyploids can possess fitness advantages over diploids.This is supported by evidence that the persistence of WGD correlates with times of environmental and climate change, suggesting potential benefit for the WGD in the face of challenges 4,35,45,[74][75][76][77] .Nevertheless, polyploids may also face substantial disadvantages, including redundant components, gene dosage imbalance, increased replication and metabolic costs, cellular mismanagement, and a higher propensity for polyploid mitosis and meiosis to produce aneuploid cells 35,58,77,78 .Despite these immediate challenges, some polyploid lineages have persisted and even thrived 79,80 .As climatic conditions stabilize and environmental conditions improve, polyploids may experience reduced fitness compared to diploids due to the accumulation of genetic load, increased mutational load, slower positive selection, and reduced growth rates 35,37,81,82 .265: 'functional analysis': do you mean GO analysis?This is unclear in the text.Describing this analysis better in the main text is needed.The results seem interesting, but they need to be better shown.
Nor do I see a clear description of this analysis in the methods.
[Reply]: The functional analyses encompass GO enrichment and gene function assessments.We have revised the sentence for clarity.For gene function assessments, we summarized key pathways from the literature and matched genes based on annotations.For GO enrichment analysis, we identified single-copy genes using the duplicate_gene_classifier module from MCScanX.We performed GO enrichment analysis of two-copy and three-copy retention groups after the WGT event with single genes as a control using BiNGO in Cytoscape (v.3.7.2).It is available in Supplementary Note.
Why is GO analysis in the Supplementary Information?In this section, our focus is on uncovering the mechanisms behind the adaptive traits of the mangrove tree, specifically waterlogging and salt tolerance.While GO enrichment analysis provides general insights, we place greater emphasis on WGT-retained genes and the pathways associated with adaptive traits.We identify specific genes and discuss them in detail within this section.The pathways of gene retention after the WGT events are more interesting and relevant, offering clearer insights than GO enrichment.This aspect is also more 1. Line 52-53.The authors concisely outline several distinct WGT events within eudicots.Notably, there are sequenced hexaploid species among eudicot plants, such as Solanum nigrum (Lee et al., 2023) and Chrysanthemum seticuspe (Nakano et al., 2021).Please add these species to the existing tree and update Supplementary Figure 1.[Reply and Revision]: We have updated Supplementary Figure 1 by incorporating three hexaploid species (Solanum nigrum, Chrysanthemum seticuspe, and Helianthus tuberosus) and annotating relevant WGT events.

7.
Line 196-211.The authors propose a model elucidating the polyploidization-rediploidization process.It seems that a cyclical nature in plant genome dynamics.It prompts consideration of the current stage of this cycle within plants.What stage are plants in now?Whether there are different

[
figure is a little bit 'light'; the novelty of the 'model' is to me not great, so I would suggest stressing the new model much, to be frank.
78: references 51 and 52 are introduced as examples or support for the statement that high quality genomes have provided opportunity to study genomes post WGD.Immediately following this statement the current study is introduced, but there is no suggestion what the gap was following studies 51 and 52 or what the major current innovation motivating this study in relation to them.It would be good to say here how the current study contributes something truly novel.

Reference 54 .
He, Z. et al.Convergent adaptation of the genomes of woody plants at the land-sea interface.Natl Sci Rev 7, 978-993 (2020).90: '204.46Mb': what was the expected size based on flow or kmers?I see these data are the methods;

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Revision]: (Line 86-89) The newly assembled genome derived from anchored contigs was 204.46 Mb, aligning closely with the genome size estimated through k-mer-based analysis (211.67 Mb).It comprised 12 chromosomes (97.60% of all sequences) and 40 unanchored scaffolds.96: 'consistent with the estimated genome size': what is that estimate?[Reply]: We have incorporated details regarding the genome size estimations of L. speciosa through both flow cytometry and k-mer-based analysis.[Revision]: (Line 92-94) The assembled genome of L. speciosa was 319.66 Mb, with an N50 value reaching 12.74 Mb, consistent with the estimated genome size (361 Mb by flow cytometry and 340.46 Mb by k-mer-based analysis).